The precise control of gene expression is essential in basic biological research as well as in biotechnological applications. Most regulated systems available in yeast enable only the overexpression of the target gene, excluding the possibility of intermediate or weak expression. Moreover, these systems are frequently toxic or depend on growth conditions. We constructed a heterologous transcription factor that overcomes these limitations. Our system is a fusion of the bacterial LexA DNA-binding protein, the human estrogen receptor (ER) and an activation domain (AD). The activity of this chimera, called LexA-ER-AD, is tightly regulated by the hormone β-estradiol. The selection of the AD proved to be crucial to avoid toxic effects and to define the range of activity that can be precisely tuned with β-estradiol. As our system is based on a heterologous DNA-binding domain, induction in different metabolic contexts is possible. Additionally, by controlling the number of LexA-binding sites in the target promoter, one can scale the expression levels up or down. Overall, our LexA-ER-AD system is a valuable tool to precisely control gene expression in different experimental contexts without toxic side effects. Regulated systems allow the adjustment of the expression of target genes by controlling a well-defined input, for example, a chemical. A regulated system should have some important features to make it applicable in multiple experimental setups. First, the system should cover a broad expression range. Second, the system should be tightly regulated to ensure no activity in absence of the input. Third, the activity of the system should not influence metabolism or be influenced by it. Fourth, the input and the activity of the system should not have any toxic effect.Saccharomyces cerevisiae. These promoters are induced by a metabolic signal via a dedicated regulator and the metabolic signal leads to the execution of a specific transcriptional program involving multiple genes (recently reviewed in (CUP1 promoter) and they require specific genetic modifications of the host to limit their negative effects  have been frequently used to control gene expression in iewed in ). By coniewed in . Some naed in CUP promoter effects . To over effects ,4, but t effects .GAL promoters and thereby influences metabolism. Finally, as the endogenous Gal4 regulator could also bind to the target promoter, this system cannot be used in media containing galactose, unless GAL4 is knocked-out  for plasmid preparation. PCR was performed with Phusion High-Fidelity DNA Polymerase , following the manufacturer's instructions. DNA manipulation was performed with restriction enzymes and T4 DNA ligase purchased from New England BioLabs, USA. The reporter and the synthetic transcription factor genes were cloned in integrative vectors derived from the pRS yeast shuttle vector series . For integration, plasmids were linearized by restriction enzyme digestion and transformed in yeast using standard methods  and induced with a concentration series of β-estradiol. During the induction, cells were cultured at 25°C and vigorously shaken  to stop translation. We incubated the samples at room temperature for 30 min in order to allow fluorescence protein maturation.TM LSRII cell analyser coupled with a high-throughput sampler (HTS) from BD Biosciences, Germany. To measure Citrine yellow fluorescence, we used a 488 nm excitation laser (100 mW) and a 530/30 nm emission filter. To measure mKate2 red fluorescence, we used a 561 nm excitation laser (100 mW) and a 610/20 nm emission filter. We used the FACSDiva software  for data recording. For each sample, we recorded 10 000 events.We subsequently analysed the samples with an LSRFortessahttp://www.bioconductor.org) to analyse the flow cytometry data. We gated for un-budded cells, which were identified by analysing the signal width of the forward scatter and the side scatter (FSC-W/SSC-W) plots as shown in Supplementary Figure S1. After gating, we obtained approximately 2000–4000 events for each sample analysed.We used the software R  with the Bioconductor package  to a final concentration of 5%.We extracted total protein, separated it by SDS-PAGE and transferred it onto a membrane as described in . When deWe used a mouse primary antibody for LexA from Dualsystems Biotech AG, Switzerland . We diluted anti-LexA 1:5000 in phosphate buffered saline + 0.1% Tween 20 (PBS-T). To detect Citrine, we used a mouse primary antibody for GFP from F. Hoffmann- La Roche Ltd, Switzerland (cat. 11814460001). We diluted anti-GFP 1:1000 in PBS-T + 0.5% bovine serum albumin . As a loading control, we detected β-actin with anti-β-actin mouse antibody from Abcam, UK  diluted 1:2000 in PBS-T. The secondary antibody was an anti-mouse from sheep linked to the horseradish peroxidase from GE Healthcare Europe GmbH, Switzerland  diluted 1:10000 in PBS-T + 1% milk .Detection was performed with ECL Detection Reagents from GE Healthcare Europe GmbH, Switzerland. We used Super RX-Films from Fujifilm AG, Switzerland, for imaging.6 cells/ml.We precultured the strains overnight at 30°C in SDC. We then diluted the cultures and grew them to a density of 1.5 × 10In each well of a 96-well plate , we inoculated 10 000 cells in 200 μL of fresh medium with a defined β-estradiol concentration. Per each strain of β-estradiol concentration combination, we prepared triplicates.We incubated the plate in the TECAN M200 reader  at 30°C for 55 h. The plate was shaken all the time. Absorbance at 600 nm was measured every 6.5 min.We plotted the growth curves using the software R. For each time point, we calculated the mean of the absorbance at 600 nm and the standard deviation of each triplicate.7 cells/ml. At time 0, we induced the culture with 2000 nM β-estradiol. At each time point, we took 2 ml from the culture, pelleted the cells by centrifugation and snap-froze the pellets in liquid nitrogen. We performed three independent biological replicates.We precultured the strain overnight at 25°C in SDC. We diluted the overnight cultures in fresh medium and grew them to a density of 1.0 × 10TM Yeast RNA Purification kit from Epicentre Biotechnologies, USA, to extract total RNA. We checked the RNA quality by agarose gel electrophoresis, as described in ® 480 Instrument using the LightCycler® 480 DNA SYBR Green I Master kit . The PCR protocol had a first denaturation step (95°C for 10 min), followed by 45 cycles of 10 s at 95°C, 10 s at 58°C and 10 s at 72°C. We measured Citrine and ACT1 cDNAs. We performed three technical replicates for each biological sample.We performed quantitative real-time PCR (qRT-PCR) on a LightCycler® 480 Software  to calculate the CP values. Relative quantification of Citrine expression in comparison to ACT1 was performed as described in  and SD-URA + 2000 nM β-estradiol plates. We incubated the plates at 30°C overnight.We developed a mechanistic dynamic model in the form of 29 ordinary differential equations (ODEs) based on the network of elementary reactions compiled in Supplementary Table S3 and on mass-action kinetics. All model simulations and analysis were performed in Matlab R2013b ; Supplementary Table S4 provides the corresponding initial conditions. Key model components are transcription factor (TF), hormone, inhibitory protein (which retains the TF in the cytoplasm when unbound to hormone), RNA polymerase II  and the corresponding mRNAs and genetic elements (operators and coding regions). Beyond binding/unbinding events and diffusive transport between nucleus and cytoplasm, gene expression and its  control are included for all components where relevant.n individual components in a biochemical network with r reactions. The general form of such an ODE system is:n x 1 vector of time-dependent concentrations c(t), the n x r stoichiometric matrix N whose entries are given by the molecularities of reaction educts and products, and the r x 1 vector function of reaction rates - or fluxes- v(?) that are calculated assuming mass-action kinetics. The fluxes depend on the system state c(t), on potentially time-varying inputs u(t) such as the β-estradiol concentration in the medium and on kinetic parameters k such as affinity constants. Finally, c0 denotes the initial state of the system, for instance, absolute protein concentrations.To generate the system of ODEs from the biochemical reaction set as given in Supplementary Table S3, we consider the mass balances of lexA boxes, where the probability of occupancy of lexA boxes, lexA boxes and (iii) a general translation rate constant In addition to mass-action kinetics, the model contains three auxiliary functions (see Supplementary Table S5 for parameter definitions): (i) a growth function  data in  and normTo map the model quantities to experimental data, we included two scaling factors (for fluorescence and mRNA abundance) as well as the following measurement models. Linear regression on variance (s.d.) as a function of average fluorescence signal from flow cytometry yielded an estimated lower detection limit of 2037 A.U.; with the corresponding slope, we assigned a variance of 2344 A.U. to all data points below this limit . To estimate variances for PCR data, we employed error propagation on the raw data. We assumed 10% measurement error for specific growth rates.lexA boxes grown in SDC . We used the training data to estimate 45 model parameters that could not be determined a priori (see Supplementary Table S4 for details). For global optimization, we employed an evolutionary strategy , cc are the calculated state variables (concentrations) of the ODE model, mc is the vector of experimental data, and σij is the corresponding measurement variance (s.d. according to the measurement models above). Simulations for the validation data used the estimated parameter values, except for condition-specific parameters. We used a χ2-test to evaluate model fit, with χ2 = 101.2, 45 estimated parameters and 401 degrees of freedom for the training data and χ2 = 860.5, 1014 d.o.f. for the validation data.The experimental data were partitioned into a training set that included only constructs with four strategy  to minimEscherichia coli genomic DNA fragments  , the hor–202 aa ,ragments ,25. The ragments . The VP1ragments .lexA boxes recognized by the LexA DNA-binding domain (see below) and controlled the expression of the yellow fluorescent protein Citrine . The target promoter contained four  Citrine . We moniP < 10−50), nor for an independent validation data set that was simulated without adjusting model parameters ; note that these statistical tests based on approximately 1400 individual data points account for the number of estimated parameters. The estimated LexA-ER-AD affinities to the general transcriptional machinery were 0.8 mM for LexA-ER-B42, 4 μM for LexA-ER-B112, 200 nM for LexA-ER-Gal4 and 27 nM for LexA-ER-VP16 (see Supplementary Table S5), well representing both the systems’ initial induction strengths and the experimentally determined affinity for the Gal4 AD alone . Specifically, we assumed that only parameters affecting the AD-RNA polymerase II interaction strength and the stability of the transcription factor protein differed between our LexA-ER-AD variants, and estimated the 45 model parameters with a (training) subset of the experimental data. The model quantitatively captured the induction dynamics in all cases Figure . OverallAD alone . We concTEF2 promoter expression levels, while LexA-ER-Gal4AD reached the ADH1 promoter  Figure . To bettACT1 promoter (Supplementary Table S1), western blotting with an anti-LexA antibody revealed different abundances of the transcription factor variants . The induction levels observed for the LexA-ER-AD variants did not directly correlate with the abundance of the transcription activator in the cells. For example, LexA-ER-Gal4AD induced moderate expression of the target gene, although it was the most abundant transcription factor. In all four variants, we detected a lower transcription factor level in the active state compared to the inactive state. This suggested a transcription-coupled degradation process  that waTo evaluate the impact of our system on cell physiology, we monitored the growth of strains containing only the synthetic transcription factor in a concentration series of β-estradiol Figure . Even atACT1 promoter and observed that its level was influenced by the activity of LexA-ER-GalAD and LexA-ER-VP16 . Strains containing these LexA-ER-AD variants showed increased red fluorescence, when incubated with more than 15 nM of β-estradiol. On the other hand, strains containing LexA-ER-B42 or LexA-ER-B112 displayed a constant mKate2 signal, when incubated in a concentration series of β-estradiol from 0 to 2000 nM. We analyzed the mKate2 levels in strains containing LexA-ER- Gal4AD or LexA-ER-VP16 over time . When induced with 15 nM of β-estradiol, these strains did not accumulate mKate2. However, when these strains were induced with higher β-estradiol concentrations, the mKate2 levels increased. We speculated that the increased red fluorescence, which reflected an accumulation of mKate2 within the cells, might be a consequence of the cell cycle slowdown caused by stress due to active LexA-ER-Gal4AD or LexA-ER-VP16. We conclude that the mKate2 levels are indeed a good proxy for the physiological status of the cells and thereby they simplify the experimental observation of small toxic effects.We asked if the constitutive expression of a fluorescence reporter gene could serve as a good proxy for the physiological status of the cell. We used the red fluorescent protein mKate2  expresseTo evaluate the induction in different growth conditions, we selected the nontoxic LexA-ER-B42 variant. We first analyzed the mRNA induction with 2000 nM β-estradiol in synthetic glucose complete minimal medium (SDC) by quantitative real time PCR (qRT-PCR). A signal appeared after 15 min, the system reached half of its maximal induction level within 45 min, and steady state was reached 1 h after induction; these dynamics were well captured by the mathematical model . We observed that steady state of the fluorescence signal was reached later compared to the mRNA . This apparent discrepancy may be explained by the delay of the fluorescence signal appearance due to intermediate steps of gene expression , protein maturation and by different stability of the species considered. We decided to use flow cytometry to evaluate the induction in different culture media. To avoid metabolic side effects, we produced a protothroph strain. We integrated empty plasmids that complemented the remaining auxotrophies of the strain carrying LexA-ER-B42 and the target gene (Supplementary Table S2). Besides SDC, which supports anaerobic growth, we tested two additional conditions. A glycerol complete minimal medium (SGlyC) supported aerobic growth. A glucose minimal medium containing proline as unique nitrogen source (SDP) mimicked nitrogen limitation.Within the first 5 h, we obtained similar induction levels in the three conditions tested. However, after 24 h, cells grown in SDP showed substantially higher yellow fluorescence than those grown in SGlyC or SDC Figure  and a weACT1 promoter controlling mKate2 to the different growth conditions , confirming that our system did not cause stress in the three conditions tested. The mKate2 levels in strains grown in SGlyc or SDP were lower compared to SDC . This reflected the adaptation of the activity of the nditions . As the lexA box, is the fragment of the bacterial lexA promoter containing two SOS motifs (lexA boxes (Supplementary Table S1). Downstream of the lexA boxes, we placed a core promoter derived from the previously characterized CYC1 promoter (CYC1 promoter region included between −68 and +16 (+1 is the most upstream transcription initiation start site), which contains two TATA elements (at −52 and −22) and an initiation region centered in +16. The TATA element at −52 controls the +16 initiation region, while the TATA element at −22 controls initiation further downstream. In our constructs, we deleted the TATA element at −22 to exclude initiation downstream of the +16 region. We termed this core promoter minimal CYC1 promoter. To insulate the synthetic target promoter from upstream transcriptional read through, we placed the DEG1 termination sequence   and the synthetic target promoter with one, two, three or four lexA boxes followed by a multi-cloning site where sequences encoding protein tags can be easily added . We tested the cassette containing four lexA boxes on URA3. As our strains displayed the ura3Δ0 deletion, we first reconstituted the wild-type locus, and then replaced the endogenous URA3 promoter with our cassette. We obtained a strain whose URA3 expression was regulated by β-estradiol . During transformation, the lexA boxes could loop out because of their repeated sequence. We checked the transformants by colony PCR and observed that only two transformants out of 24 checked had less than four lexA boxes.To make our induction system more accessible to the yeast community, we constructed a set of cassette plasmids for PCR-based promoter replacement (Supplementary Table S1). These plasmids contain an antibiotic resistance (Saccharomyces cerevisiae. In contrast to most efforts in the field, we employed variants of ADs, and, combined with a mathematical model, we demonstrated that this design parameter offers currently under-used possibilities for engineering transcription control systems with quantitatively predictable performance. Our LexA-ER-AD is a modification of the widely used Gal4-ER-VP16, and it overcomes intrinsic limitations of Gal4-ER-VP16. A crucial result of our work is the implementation of a nontoxic transcription factor. We showed that the AD could have a role in the toxic phenotype frequently observed in synthetic expression systems such as Gal4-ER-VP16 (We engineered the LexA-ER-AD expression system to precisely control transcription in a hormone-dependent manner in -ER-VP16 . LexA-ERBesides avoiding toxicity, we also wanted a system that is controllable irrespective of the cell's physiological state. We therefore substituted the Gal4 DNA-binding domain with the bacterial LexA. Theoretically, transcription factors with a heterologous DNA-binding domain do not target sites in the host genome and do not require genetic modifications of the host strain for proper activity in different physiological contexts. Reduction of the off-targets of the heterologous transcription factor also results in reduced toxicity: transcription factors based on artificial zinc-finger DNA-binding domains do not have any off-targets and they do not inhibit growth, even when they contain the VP16 AD . By keepTDH3 promoter, one of the strongest promoters known in yeast (ADH1 and TEF2 promoters (We constructed our LexA-ER-AD system to precisely adjust the expression of the target gene to low, intermediate or high levels, rather than for overexpression. While LexA-ER-Gal4AD and LexA-ER-VP16 caused a steep graded response, the less steep induction with LexA-ER-B42 and LexA-ER-B112 allowed a finer and more precise regulation of the target gene. We classified the variants containing B112 and VP16 as strong, since their maximal induction levels mapped in the range of the in yeast ,40. We dromoters ,10.lexA boxes in each target promoter. In general, the strength of the target promoter can also be modulated by modifying the affinity of the DNA-binding domain to its target sites (lexA boxes in the target promoter, we expect to be able to precisely control all possible levels of gene expression with our inducible system.The structure of the target promoter represents an additional layer for gene expression regulation. By modifying the number of transcription factor-binding sites, it is possible to adjust the strength of the induction ,41. Withet sites ,41. HoweSupplementary Data are available at NAR Online.


We aimed to develop a cost-effective and robust method to predict drug resistance in individual patients. Representative tissue fragments were obtained from tumors removed from female patients, aged 24-74 years old. The tumor tissue was taken by a histopathology’s or a surgeon under sterile conditions. Cells obtained by enzymatic dissociation from tumor after surgery, were cultured as a monolayer for 6 days. Paclitaxel, doxorubicin, carboplatin and endoxan alone or in combination were added at the beginning of culture and after 6 days, Alamar blue test was used for showing action on cell proliferation why caspase- 3 activity assays for verifying action on apoptosis. Inhibitory action on cell proliferation was noted in 2 of 12 patients tumor treated with both single and combined drugs. Using caspase-3 assay we showed that 50% of tumor cells was resistant to single chemotherapeutic drugs and 40% for combined. In 2 of 12 tumors, which did not reacted on single drugs, positive synergistic action on cell proliferation was observed in combination of D + E and C + E. This pilot study suggests: 1) monolayer culture of tumor cells, derived from individual patients, before chemotherapy could provide a suitable model for studying resistance for drugs; 2) caspase-3 activity is cheap and useful methods; 3) Alamar blue test should be taken into consideration for measuring cell proliferation. Ovarian cancer is the most lethal gynecologic malignancy in the developed world , and theHowever, advanced ovarian cancer typically becomes chemo-refractory within approximately 2 years, and second-line treatment options do not provide a significant survival advantage -6. ThereStudies in cell lines do not always correlate well with results from tumor tissue, which may consist largely of non-neoplastic cells that support and modify the biology of neoplastic cells . TherefoThe ability of a cancer cell to respond to a chemotherapeutic agent is believed to be due, in part, to its apoptotic capacity . In the Material and methodsReagentsHams F-10 medium, collagenase type H, antibiotic-antimycotic solution (100 ×), fetal bovine serum  and trypan blue were obtained from Sigma Chemical Co. . Phosphate-buffered saline (PBS) was purchased from the Laboratory of Sera and Vaccines . Paclitaxel, doxorubicin, carboplatin and endoxan were obtained from the Oncological Surgery Department of the National Cancer Institute, Cracow Branch, and Poland.Tumors samples et al. , doxorubicin (2.5µM), carboplatin (13.4µM), or endoxan (19µM), either alone or in the following combinations: 1) paclitaxel plus doxorubicin; 2) paclitaxel plus carboplatin; 3) paclitaxel plus endoxan; 4) doxorubicin plus carboplatin; 5) doxorubicin plus endoxan; or 6) carboplatin plus endoxan. Drug doses were based on those of Nicolantonio et al.  at 4°C. Ovarian tumor tissue was cut into small pieces and placed in Petri dishes containing PBS. Cells were obtained by enzymatic dissociation, primarily using 0.75mg/mL collagenase (Sigma Cat No. C-8051), repeated three times for 15 min at 37°C. Cells were then assessed for viability by trypan blue exclusion. Approximately 20,000–30,000 cells/well were added to each well of a 96-well plate with 200µL/well 10% (FBS) medium. Plates were then incubated at 37°C in 5% COclitaxel .8µM, doxMeasurement of cell proliferation using Alamar Blue Alamar Blue assay  is designed to measure quantitatively the proliferation of various human and animals cell lines based on detection of metabolic activity. The active ingredient is resazurin (IUPAC name: 7-hydroxy-10-oxidophenoxazin-10-ium-3-one), also known as diazo-resorcinol, azoresorcin, resazoin, resazurine, which is water-soluble, stable in culture medium, is non-toxic and permeable through cell 50 years to assess cell viability and cytotoxicity in a range of biological and environmental systems and in a number of cell types including bacteria, yeast, fungi, protozoa and cultured mammalian and piscine cells. It offers several advantages over other metabolic indicators and other cytotoxicity assays , 15. TheMeasurement of apoptosis by caspase-3 activityThe caspase-3 colorimetric assay is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p- nitroanilide (Ac-DEVD-pNa) by caspases-3, resulting in the release p-nitroaniline (pNA) moiety. The concentration of the pNA released from the substrate is calculated from the absorbance values at 405 nm. The comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the fold increase in caspase-3 activity. After replacing media with caspase assay buffer , cell lysates were incubated with a caspase-3 colorimetric substrate, acetyl-Asp-Glu-Val-Asp-p-nitroanilide-7-amino-4-methylcoumarin (Ac-DEVD-AMC conjugation); Sigma). Reaction mixtures were incubated at 37°C for 15 min to 1h, and the absorbance was measured at 405nm using an ELISA microplate reader (BioTek Instruments). Caspase 3, which is an effector caspase, is the most studied of mammalian caspases. Caspase 3 plays a central role in mediating nuclear apoptosis including chromatin condensation and DNA fragmentation as well as cell blebbing. Caspase 3 activity is tissue, cell type, or death stimulus specific. Statistical analysis Each treatment was repeated three times (n = 3) in quadruplicates, and the average of the values was used for statistical calculations. Statistical analysis was performed using Stastistica 6.0 . Data were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey honestly significant difference (HSD) multiple range test. In the presented data we used monolayer cell cultures obtained from individual patients. Cells were exposed for 6 days to individual or combined chemotherapeutic drugs. It has been showed that 80% of the tumor cells were resistant to chemotherapeutic drugs. We observed inhibition of cell proliferation in two of the 12 tumor cultures treated with paclitaxel, carboplatin or doxorubicin using Alamar Blue test, and only one of the 12 cultures treated with endoxan . Four ofApoptosis is programmed cell death that involves the controlled dismantling of intracellular components while avoiding inflammation and damage to surrounding cells. Apoptosis is a mode of programmed cell death that is coordinated by members of the caspases family of cysteine proteases. The caspase-3 plays a central role in the execution-phase of cell apoptosis. Several attempts have been made within the last decade to develop molecules capable of directly activating caspase-3 for use in cancer therapy. A particular target suggested for intervention has been the “safety catch” sequence present in inactive procaspase-3 . Using cThe results presented are in agreement with previously published data. Chemotherapeutic drug resistance is a common feature of ovarian cancer and is a major problem given that the majority of EOCs are in an advanced stage when a diagnosis is made . The staet al. ( In clinical trials in 1989, paclitaxel was reported to produce partial or complete responses in 30% of patients with advanced ovarian cancer , 21. Di et al.  have shoet al. ( et al. (van der Burg et al. (Tan et al. (Our data are also in agreement with recent published data by Pitakkarnkul et al. , which s( et al.  evaluate( et al. . A receng et al.  demonstrn et al.  recentlyin-vitro assay will really predictive of response.Activation of the caspase-3 pathway is a hallmark of apoptosis and can be used in cellular assays to quantify activators and inhibitors of the “death cascade. Our data suggest that simple, monolayer cultures of tumor cells and caspase-3 assays as a key biomarker for apoptosis are a relatively cheap and fast method taken before chemotherapy. This representative apoptosis assay can be used in an automated way and is amenable to development of high-throughput applications. However, the effective response of the patients, from whom cell cultures were derived, to front line chemotherapy and if the


The Physician Global Assessment (PGA) is an important and useful outcome measurement of lupus disease activity. Consensus on when the PGA should be performed is lacking; many lupus specialists determine the PGA at the time of a patient visit while others determine the PGA after receipt of laboratory values. The objective of this study was to collect preliminary data to determine the optimal time to perform a PGA.t test was utilized to compare the pre-laboratory PGA with the post-laboratory PGA.In this pilot study, a PGA was performed by a single clinician upon completion of an outpatient clinical encounter and again after receipt of pertinent laboratory values. The pre-laboratory PGA was determined with knowledge of the subject's previous laboratory values and lupus history, while the post-laboratory PGA was determined after laboratory reports from the patient clinical encounter were also available. Disease activity could range from 0 to 3, with 0 representing no disease activity, 1 representing mild disease activity, 2 representing moderate disease activity and 3 representing the most severe lupus disease imaginable. Laboratory values obtained at each clinical visit included a CBC, comprehensive chemistries, C3, C4, anti-dsDNA, urinalysis and, if pertinent, a spot urinary protein/creatinine ratio. Disease duration and SELENA SLEDAI were recorded. Results are presented using descriptive statistics; the paired Student's P = 0.02 paired Student's t test). Twenty-six pre-laboratory PGAs were evaluated as 0, 23 remained at 0 after receipt of laboratory examinations, and three increased secondary to new lupus serologic activity. The pre-laboratory and post-laboratory PGAs were equal in 17 of 53 encounters with disease activity . The average pre-laboratory PGA of these 53 encounters was 0.67, the average post-laboratory PGA was 0.77  and the average SELENA SLEDAI was 3.12 (range 0 to 8).Thirty-three patients, three male and 30 female with an average SLE disease duration of 12.3 (range 1 to 36) years, contributed 74 assessments to this study. The average SELENA SLEDAI was 2.21. The average pre-laboratory PGA was 0.45 and the average post-laboratory PGA was 0.55 (In some lupus patients, the PGA determined prior to receipt of laboratory values may be the same as the PGA determined after labs are received. However, in these preliminary data, there was a significant difference between pre-laboratory and post-laboratory PGA. Further studies in a larger patient population are needed to confirm and extend these findings.


Both PRP and SF-MSCs could be feasibly used in regenerative medicine and orthopedic surgeries.This article studied the effects of platelet-rich plasma (PRP) on the potential of synovial fluid mesenchymal stem cells (SF-MSCs) to differentiate. The PRP and SF-MSCs were obtained from the blood and knees of pigs, respectively. The identification of SF-MSCs and their ability to differentiate were studied by histological and surface epitopes, respectively. The SF-MSCs can undergo trilineage mesenchymal differentiation under osteogenic, chondrogenic, and adipocyte induction. The effects of various PRP concentrations  on differentiation were evaluated using the SF-MSCs-alginate system, such as gene expression and DNA proliferation. A 50% PRP concentration yielded better differentiation than the 20% PRP concentration. PRP favored the chondrogenesis of SF-MSCs over their osteogenesis in a manner that depended on the ratios of type II collagen/type I collagen and aggrecan/osteopontin. Eventually, PRP promoted the proliferation of SF-MSCs and induced chondrogenic differentiation of SF-MSCs  Both mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) have attracted a lot of attention in recent years because of their potential use in regenerative medicine and tissue engineering. MSCs are pluripotent cells that have the capacity for trilineage mesenchymal differentiation in response to appropriate signals . MSCs haSynovial fluid mesenchymal stem cells (SF-MSCs) are MSCs that are present in synovial fluid. Owing to their location, harvesting SF-MSCs is less invasive than harvesting other MSCs, such as bone marrow (BM), umbilical cord blood, and adipose tissue. Osteoarthritis ,8, anterPRP contains concentrated growth factors, including transforming growth factor beta (TGF-β), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) ,12,13. Tin vitro.Both SF-MSCs and PRP have the advantage of being autologous and easy to harvest, and they have been observed to have positive effects in clinical practice. However, the exact mechanisms how SF-MSCs and PRP work are not yet understood. Therefore, this study looks at the effects of PRP on SF-MSCs Spindle-shaped SF-MSCs of pigs were observed using a light microscope; their phenotype is similar to that of human SF-MSCs . Only a Positive rates for cluster of differentiation 29 (CD 29), CD 44, and CD 90 in the SF-MSC population were close to or exceeded 90%. CD 105-positive rates in the same population exceeded 60%, whereas CD 34- and CD 45-positive rates were less than 5% . In concp < 0.05). The expression of all marker genes by SF-MSCs exceeded that by BM-MSCs  and adipocyte protein 2 (aP2) for adipogenesis), which were conducted in induction media. The results thus obtained reveal that all marker genes were upregulated in induction media during a one-to-three week cultivation period relative to the control group (cultivated without induction media)  hydrogel system was utilized to evaluate cultivation by three PRP concentrations .The p < 0.05). This result reveals that PRP promoted SF-MSCs’ proliferation, which is consistent with a previous report [As displayed in s report .p < 0.05). The expression levels in all gene profiles, except for that of osteopontin, increased dose-dependently with PRP concentration during the cultivation period (p < 0.05). The 50% PRP concentration promoted differentiation more than did 20% PRP (p < 0.05). To confirm the effects of PRP on SF-MSCs’ differentiation, ratios of chondrogenesis-related genes to osteogenesis-related genes (type II collagen/type I collagen and aggrecan/osteopontin) were compared . Additionally, the concentration of TGF-β1 exceeded that of bFGF (p < 0.05). According to the literature, bFGF, TGF-β1, and PDGF are associated with the osteogenesis, chondrogenesis, and proliferation of MSCs, respectively [The concentrations of growth factor concentrations in PSA hydrogels (20% and 50%) were evaluated. The PRP in the PSA hydrogel system herein was activated using calcium ions to promote the release of growth factors ,24. The ectively ,26,27,28et al. isolated synovial MSCs from human synovial membrane for the first time [et al. support this idea as they found a correlation between the presence of synovium clumps and the number of SF-MSCs [Independently of their origin, MSCs have both high proliferative potential and a multipotentiality for chondrogenic, osteogenic and adipogenic differentiation . In 2001rst time . As wellrst time . SF-MSCsrst time ,10. Jone SF-MSCs . The preet al., SF-MSCs in patients with an injured anterior cruciate ligament were 100 times more abundant than in healthy volunteers [et al. identified SF-MSCs in normal human and bovine synovial fluid from the knee joint, and found that the number of SF-MSCs was significantly increased in a knee with osteoarthritis [et al. further showed that the number of SF-MSCs increased with the radiological osteoarthritis grade [Although the exact role of SF-MSCs in differential potential is not yet clear, SF-MSCs are thought to participate in intra-articular healing. According to Morito rthritis . Sekiya is grade . In an ais grade ,32.PRP is an emerging biological tool in orthopedics and has been utilized clinically in the last decade. Percutaneous injection of PRP has successfully relived pain associated with chronic epicondylitis ,16 and cin vivo applications, the hydrogel system was used to eliminate the possibility of their being easily washed out with liquid and of the quick release of growth factors following activation by calcium. Our earlier study proved that SF-MSCs with PRP and thermosensitive hydrogel were very effective in cartilage regeneration and maturation. As published results have shown, 10% PRP in thermosensitive hydrogel can promote the repair of defective cartilage [in vitro experiments were conducted to explain the role of PRP in cartilage defect repair and to determine whether the effects are related to the concentration of PRP. As shown in et al. [Owing to the favorable results of various clinical applications of PRP and the positive effects of SF-MSCs in many studies, the effect of PRP on SF-MSCs was evaluated herein. With an eye to future artilage . Howevern et al. , who foun et al. ,27, bFGFn et al. , and PDGn et al. . Based oAll procedures performed on small-eared pigs were conducted according to the Guide for the Care and Use of Laboratory Animals and approved by the Committee of Experimental Animal Sciences of Chang Gung Memorial Hospital . Female, six small-eared pigs (weight 25 to 35 kg) were anesthetized with an intramuscular injection of a mixture of 1 mL of Rompun and 10 mL of ketamine. Ten milliliters of saline was injected into the knee joint with an 18G needle and 20-mL syringe, and then arthrocentesis was performed with the 18G needle and 20-mL syringe 30 s later. Under sterilized conditions, the combination of synovial fluid and saline was centrifuged at 1500 rpm for 5 min to separate the supernatant, and the pelleted cells were resuspended in Dulbecco Modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 1% antibiotics/antimycotics . The culture medium was replenished twice a week. The cell morphology was observed with a light microscope. The passage 4 of SF-MSCs was used in each experiment in this study.All procedures performed on small-eared pigs were conducted according to the Guide for the Care and Use of Laboratory Animals and approved by the Committee of Experimental Animal Sciences of Chang Gung Memorial Hospital. Female, six small-eared pigs (weight 25 to 35 kg) were anesthetized with an intramuscular injection of a mixture of 1 mL of Rompun and 10 mL of ketamine. Ten milliliters of marrow were aspirated from the tibia using an 18-gauge needle, mixed with 1 mL of heparin (3000 units per mL)  in a 15-mL centrifugation tube, and then washed with phosphate buffered saline (PBS) twice and the pelleted cells resuspended in DMEM supplemented with 10% FBS and 1% antibiotics/antimycotics . The culture medium was replenished twice a week. The passage 4 of MSCs was used in this study.+, CD 34−, CD 90+, CD 45−, CD 44+, and CD 105+ [For epitope profiling of the SF-MSCs, one million cells were suspended in 500 μL of PBS containing 20 μg/mL of antibody. After incubation for 30 min at 4 °C, cells were washed with PBS. Fluorescein isothiocyanate- or phycoerythrin-coupled antibodies against CD29, CD34, CD90, CD45, CD44, and CD105  were used. The SF-MSCs were supposed to express as the followings, CD 298 platelets/mL, and that in PRP was (1.9 ± 0.6) × 109 platelets/mL (422% more in PRP than in whole blood).The PRP was collected by drawing whole blood from small-eared pigs via neck vessel penetration. Using a syringe with an 18-gauge needle, 9.8 mL of blood were aspirated and mixed with 0.2 mL of sterilized sodium citrate  for anticoagulation. The whole blood was then centrifuged at 1200 rpm  for 10 min, and the superstratum formed the PRP. The numbers of platelets in the PRP and in the whole blood were counted respectively by a hemocytometer under a light microscope. The concentration of platelets in whole blood was (4.5 ± 0.8) × 10in vitro multidifferentiation potential of the SF-MSCs and BM-MSCs toward osteogenesis, chondrogenesis, and adipogenesis was evaluated [−7 M dexamethasone, 50 g/mL ascorbate-2-phosphate, 40 g/mL proline, 100 g/mL pyruvate, and 50 mg/mL Insulin-Transferrin-sodium selenite (Sigma). Adipogenic differentiation was induced in expanded MSCs cultures by treatment with 1-methyl-3-isobutylxanthine, dexamethasone, insulin, and indomethacin.The valuated . OsteogeMacrocystitis pyrifera, has a mannuronic acid/guluronic acid ratio of 1.67 and a molecular weight of around 50,000 Dalton. One milliliter of alginate (10%) was dissolved in 9 mL of PBS and sterilized for 30 min at 121 °C in an autoclave, then cooled at room temperature. PRP was added to the 1% alginate-PBS solution to form PRP-alginate solutions with 20% and 50% concentration of PRP, respectively, in a laminar flow hood. The SF-MSCs were treated with 0.5% trypsin before four passages, and a 5 × 104 cells/mL density was prepared in the PRP-alginate solution. In addition, SF-MSCs were added into the alginate-PBS solution without PRP to make the control group. Then the PRP-SF-MSCs-alginate solution was dropped into a 0.1 M calcium chloride solution. After 2 min of reaction, the PSA hydrogel was obtained  was used in these studies. This alginate is derived from obtained A,B. ThenThe cultured cells in the monolayer dish or PRP alginate hydrogels with the chondrogenic and osteogenic medium were analyzed by real-time PCR to investigate temporal mRNA expression changes. Total RNA was extracted from the cultured cells inside the monolayer dish for 1-week, 2-week, and 3-week cultivation, or PRP alginate hydrogels for 2-week cultivation. The monolayer dish or PRP alginate hydrogels were incubated with TRIzol reagent . Subsequent steps were followed according to the manufacturer’s instructions. The total RNA of each sample was measured using a spectrophotometer  and then was reverse transcribed with oligo-(dT) primers using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase from a First Strand cDNA Synthesis Kit . The resultant solution (2.0 μL) was amplified in triplicate by real-time PCR  with SyBr Green Master Mix reagent (Applied Biosystems). Glyceraldehyde phosphate dehydrogenase (GAPDH) was also amplified under the same conditions as a control. The primers for the specific genes were designed according to the published sequences available in GenBank using Primer Express 2.0 (Applied Biosystems) as shown in 2 incubator at 37 °C. Consequently, the formed formazan crystal was dissolved by adding 2 mL of MTT solubilization solution. The solution was then transferred into 1.5-mL cuvettes, and the absorbance at 570 nm was taken three times.Cell proliferation was measured by MTT assays on days 0, 7, and 14 . Briefly, SF-MSCs in PRP alginate hydrogels were cultivated in 96 wells first, then 2 mL of medium containing 0.5 mg/mL MTT was added into each culture well and incubated for 4 h in a 5% COAfter PRP was harvested, the TGF-β1 (ng/mL), bFGF (ng/mL), and PDGF were evaluated by Porcine TGF-β1 Elisa kit , Porcine bFGF Elisa kit (CUSABIO), and Porcine PDGF Elisa kit (CUSABIO) according to the manufacturer’s protocols. Briefly, 100 μL of sample and standard were added to each well for 1-h incubation at room temperature. After washing three times, 100 μL of the working dilution of the detection antibody was added to each well for 1-h incubation. After washing three times again, 100 μL of the working dilution of detection agent was added to each well for incubation for 20 min. Finally, 100 μL of the substrate solution was added to each well for 5-min incubation and then stop solution was added. The optical density of each well was taken immediately, using an enzyme-linked immunosorbent assay (ELISA) reader set to 450 nm. The values of TGF-β1, bFGF, and PDGF were shown in i.e., Alizarin red S staining, Alcian blue staining, and Oil red O staining, are according to the standard protocols. Briefly, SF-MSCs cultivated in chamber slides were fixed in 10% neutral buffered formalin. Alizarin red S staining was used to identify calcium deposition for osteogenesis. Alcian blue staining (Sigma) was used for proteoglycans detection for chondrogenesis. Oil red O staining was used to identify lipids and fats for adipogenesis. For immunofluorescence staining, SFMSCs in the PSA hydrogel was fixed in 10% neutral buffered formalin and embedded in paraffin. Then the sections of 5 μm were cut and collected on slides. Following permeabilization with 0.2% Triton X-100  and blocking solution treatment (5% non-fat milk in PBS with 0.1% Triton X-100) for 30 min at room temperature, the slides were incubated with mouse anti-porcine monoclonal primary antibodies of type I collagen, type II collagen, and PPAγ2 , applied for 1.5 h at room temperature. Subsequent to being washed three times with 1% non-fat milk in PBS with 0.1% Triton X-100, the slides were treated with Alexa 488 anti-mouse immunoglobulin G1 secondary antibody  for 45 min at room temperature. Finally, the samples were mounted using a mounting solution with DAPI  and left to dry overnight prior to observation.The detailed protocols of histological staining, n = 3) for each test. The control and experimental groups were compared to each other by a t-test.Each experiment was replicated three times. The data were expressed as means ± standard deviation (SD) by three experiments (in vitro. All of these make PRP and SF-MSCs potential tools in regenerative medicine and orthopedic surgeries.In conclusion, both PRP and SF-MSCs have the advantage of being autologous and easier to harvest. Compared with recombinant growth factors or other products from animals, PRP is safer in terms of immunological reaction and less expensive. In this study, we found that PRP could enhance proliferation of SF-MSCs and induce chondrogenic differentiation of SF-MSCs


Reliability of the Actigraph GT3X+ accelerometer has not been determined under normal wear time criteria in a large sample of subjects and accelerometer units. The aim of this study was to assess contralateral hip difference and inter-instrument reliability of the Actigraph GT3X+ monitor in adults under long-term free-living conditions.2) concurrently wore two GT3X+ accelerometers  attached to contralateral hips for 21 days. Reliability was assessed using Bland-Altman plots, mixed model regression analyses and absolute measures of agreement for different lengths of data accumulation .Eighty-seven adult subjects  age 31.3 (12.2) years; body mass index 23.7 (3.1) kg/mThere were no significant differences between contralateral hips . Inter-instrument reliability increased with increased length of data-accumulation. For a 7-day measurement period (n = 232 weeks), limits of agreement were ±68 cpm  and ±81.3 cpm (vector magnitude) for overall physical activity (PA) level, ±51 min for sedentary time, ±18.2 min for light PA, ±6.3 min for moderate PA, ±3.5 min for vigorous PA, and ±6.7 min for moderate-to-vigorous PA.The Actigraph GT3X+ accelerometer is a reliable tool for measuring PA in adults under free-living conditions using normal data-reduction criteria. Contralateral hip differences are very small. We suggest accelerometers be attached to the right hip and data to be accumulated over several days of measurement. Despite some limitations, there are general agreement that accelerometry provide valid estimates of free-living physical activity (PA) and sedentary behavior (SED) in diverse study settings. Yet, as reliability is a premise for validity , determiIntra- and inter-instrument reliability has been studied for a wide range of accelerometer makes and models using a range of study set-ups. Of these monitors, the Actigraph monitors  and Manufacture Technology Incorporated (MTI) models) are the most used and studied, and have shown favorable reliability compared to other monitor brands –5. The eIn 2009/2010 Actigraph released the tri-axial accelerometer GT3X/GT3X+ –14, of wThe current studies that have investigated the inter-instrument reliability of the GT3X/GT3X+ monitor have several critical limitations. First, findings are based on relatively few subjects and/or monitors, which question the validity and generalizability of their findings. Secondly, free-living and intensity-specific PA have only been investigated in a single study  using a The aim of the present study was to assess the contralateral hip difference and the inter-instrument reliability of the Actigraph GT3X+ monitor to determine overall PA level as well as intensity-specific PA and SED in adults under free-living conditions. We included a 21-day measurement period in 87 subjects and applied a total of 174 accelerometer units. We hypothesized that the inter-instrument reliability would improve along with an increased length of the measurement period, and that there would be no difference between contralateral hips.Eighty-seven subjects were recruited by word of mouth mainly among students and staff at the Sogn og Fjordane University College, Norway for a long-term (21 days) objective measurement of PA level. Students and staff at the Sogn og Fjordane University College received oral and written information regarding the study. Accelerometers were thereafter made available for pick-up and later returned at Campus, using a “mail-box”. Thus, the data was collected anonymously. The study was reviewed by the Regional Committee for Medical and Health Research Ethics of Western Norway, who confirmed that their approval was not required.Physical activity was measured using a total of 174 Actigraph GT3X+ accelerometers (firmware 2.2.1) . Two accelerometer units were attached to an elastic belt, and worn at contralateral hips over a 21-day period. Half-way through the measurement period (day 11), subjects switched the accelerometer units around . Thus, each accelerometer unit was worn at both hips. This procedure allowed for an accurate analysis of differences between hips, as we avoid confusion of differences between hips and differences between accelerometer units. Subjects were instructed to wear the accelerometers at all times, except during water activities  or while sleeping. The accelerometers were initialized at a sampling rate of 30 Hz. Files were analyzed at 10 second epochs using the Kinesoft v. 3.3.75 software . A wear 2).Subject characteristics  were self-reported. Body mass index (BMI) was calculated as the body mass divided by the squared height (kg/mSubject characteristics were reported as means and standard deviations (SD).Agreement was assessed by determining a) the mean difference (bias) between contralateral hips and b) the measurement error between the paired accelerometer instruments. The difference between the contralateral hips was analyzed using a linear mixed model regression analysis including a random intercept for subjects, including a dummy variable indicating a) the right versus the left hip and b) the dominant versus the non-dominant hip in separate analyses. Day 11 was excluded from these analyses, leaving a total of 2711 days for analysis across subjects and accelerometer units. Residuals were normally distributed in all models. Findings were reported as regression coefficients and 95% confidence intervals (CI), along with effect sizes (estimated difference/pooled SD).The inter-instrument agreement  was analyzed using three different time intervals for accumulating data: 1) Single days of measurement; 2) Three 7-day periods of measurement; 3) The total 21-day period. Thus, in the first and second analysis, up to 3 weeks and 21 days of measurement were included for each subject, respectively. Error was determined using Bland Altman plots, showing the difference between two accelerometer units as a function of the mean of the two units , and by All analyses were performed using IBM SPSS v. 20 . A p-value < .05 indicated statistically significant findings.2) was included in the analyses.All 87 subjects (28 (32%) men and 59 women; mean (SD) age 31.3 (12.2) years; body mass 70.4 (12.2) kg, height 172.1 (8.1) cm, body mass index 23.7 (3.1) kg/mIn total, 1397 (76%) days, 232 (89%) weeks and 80 (92%) 3-week periods were valid according to the criteria applied, and included in the analyses. Evaluation of differences between contralateral hips (right (n = 1361 days)–left (n = 1350 days) showed very small (ESs <0.042) and no statistically significant differences . DiffereThe correlation between accelerometer units 1 and 2 were generally very high . As shown in The present study determined the inter-instrument reliability for the measurement of free-living PA and SED over 21 days using 174 Actigraph GT3X+ accelerometer units in 87 subjects. Thus, to the best of our knowledge, it is the first study that has investigated the inter-instrument reliability of many GT3X+ accelerometer units under normal conditions of measurement  in a relatively large sample. For a 7-day measurement period, output from accelerometer units differed by (95% LoA) ±68 CPM, ±51 minutes of SED and ±6.7 minutes of MVPA. Inter-instrument reliability decreased for a shorter period of measurement, and improved when measurements were accumulated over a longer time-frame.Contrary to our findings, significant differences in accelerometer output between contralateral hips have been detected in some previous studies , 13, butThe clear pattern of improved inter-instrument reliability along with increased length of data accumulation found in the present study supported the hypothesis put forward. To the best of our knowledge, we are the first to report findings on inter-instrument reliability having measurements over periods longer than 24 hours. This is an important finding, as inclusion of ≥3–4 days of valid wear time is recommended to obtain valid results from accelerometer measurements . InteresThe improved reliability with increased accumulation of data is in accordance with probability theory and the law of large numbers, that is, the average result will tend to be closer to the expected value when the number of observations increases. Specifically, as there is a certain amount of intra-unit variation over time –7, the sPrevious studies assessing free-living intensity-specific PA and SED have detected variation in inter-instrument reliability over different intensity-categories. The greatest variation has been shown for SED (CV 10.5%) , MPA CV where a To arrive at a decision whether a measure is reliable and fulfills analytical goals for a given study is a difficult case. Although some seems to choose some arbitrary cut point for acceptable reliability , a measure cannot be “significantly reliable”, thus leaving this decision to a matter of opinion and judgment , 22. As The present study has several strengths. First, we included a large sample of subjects and monitors compared to previous studies. Thus, results are likely to be generalizable to a normal adult population and to the general pool of Actigraph GT3X+ accelerometers. Second, whereas previous studies investigating inter-instrument reliability under free-living conditions were limited to 24 hour monitoring periods, subjects in the present study wore the accelerometers for a long period (21 days). As our findings clearly showed that the inter-instrument reliability increased along with increased time of monitoring, the study provides researchers important insight into the relationship between monitoring length and reliability. Third, the comparison of contralateral hips was performed using a within-accelerometer controlled design. Thus, we were able to determine differences between hips per se.Possible limitations to the present study include the use of multiple observations from the same subjects in analyses of single-day- and 7-day periods of measurement, and the inclusion of an adult sample. We used mixed-model analyses to account for clustering (adjustment of standard errors) when determining effect-estimates for differences between contralateral hips. Such a procedure is not straight-forward for reliability-analyses of random errors, where individual observations are modeled (as opposed to group means). Evaluation of the dependency of observations revealed low levels of clustering (intra-class correlation (ICC) <3.5% for single-day- and ICC <5.8% for 7-day periods of measurement). Inclusion of a random effect for subjects was not indicated for the 7-day measurements, as evaluated using the likelihood ratio test . For sinThe Actigraph GT3X+ accelerometer is a reliable tool for measuring overall PA level and intensity-specific PA and SED in adults under free-living conditions using normal data-reduction criteria. Inter-instrument reliability increased when accelerometer data were accumulated over a longer time-period. Furthermore, contralateral hip differences were minimal. Although inter-instrument differences are minor, the differences between units should be recognized, because noise in any measurement will attenuate “real” relationships between PA and health and increase the likelihood of performing type II errors. Along with current recommendations, we suggest accelerometers be attached to the right hip for consistency and data to be accumulated over several days of measurement.S1 Dataset(XLSX)Click here for additional data file.


Clostridium tyrobutyricum CIP I-776 (IFP923), an efficient producer of butyric acid. The genome consists of a single chromosome of 3.19 Mb and provides useful data concerning the metabolic capacities of the strain.Here, we report the draft genome sequence of  Clostridium species  and sequenced on an Illumina MiSeq with a 2 × 250-bp paired-end sequencing kit. A total of 3,756,334 reads were assembled using the Velvet assembler :acetoacyl-CoA transferase were detected. These genes do not have homologues in C. acetobutylicum and may have a key role in the conversion of butyryl-CoA to butyrate in C. tyrobutyricum, consistent with observations made by other authors (While one  authors . FurtherFAXL00000000. The version described in this paper is the first version, FAXL01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Wnt and Notch signaling pathways are critically involved in relative cell fate decisions within the development of cutaneous tissues. Moreover, several studies identified the above two pathways as having a significant role during wound healing. However, their biological effects during cutaneous tissues repair are unclear.We employed a self-controlled model (Sprague–Dawley rats with full-thickness skin wounds) to observe the action and effect of Wnt/β-catenin and Notch signalings in vivo. The quality of wound repair relevant to the gain/loss-of-function Wnt/β-catenin and Notch activation was estimated by hematoxylin-and-eosin and Masson staining. Immunofluorescence analysis and Western blot analysis were used to elucidate the underlying mechanism of the regulation of Wnt and Notch signaling pathways in wound healing. Meanwhile, epidermal stem cells (ESCs) were cultured in keratinocyte serum-free medium with Jaggedl or in DAPT acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl) to investigate whether the interruption of Notch signaling contributes to the expression of Wnt/β-catenin signaling.c-Myc and Hes1, which ultimately lead to enhanced or delayed wound healing. Furthermore, Western blot analysis suggested that the two pathways might interact in vivo and in vitro.The results showed that in vivo the gain-of-function Wnt/β-catenin and Notch activation extended the ability to promote wound closure. We further determined that activation or inhibition of Wnt signaling and Notch signaling can affect the proliferation of ESCs, the differentiation and migration of keratinocytes, and follicle regeneration by targeting c-Myc and Hes1 separately. What’s more, interaction between the above two pathways might act as a vital role in regulation of wound healing.These results suggest that Wnt and Notch signalings play important roles in cutaneous repair by targeting  The skin is the largest organ in the human body and stems aggression of external microorganisms and dehydration. As a response to and result of injury, several dynamic and interactive processes occur and eventually lead to wound healing, which involves regeneration of the normal structure and function of the organ. The success of the wound repair depends on the differentiation and proliferation of involved cells, including epidermal stem cells (ESCs), keratinocytes, and fibroblasts, together with the assistance of various biological signals. Moreover, these signals contribute significantly to regulate biological actions of cells within epithelial tissue. Therefore, under the incorrect guidance of signals, activities of the above cells change and the resulting wound healing is abnormal . According to increasing advances in wound-healing research, Wnt and Notch signaling pathways play a key role in the regulation of migration, proliferation, and differentiation of cells functionally relevant to skin tissue repair.c-Myc). Wnts are important for many fundamental cellular processes in the regulation of development and homeostasis  × 100 %  and Masson. The skin tissue was examined in random order under blindfold conditions with standard light microscopy.Hes1 antibody and rabbit anti-c-Myc antibody . The samples were then incubated for 1 h in 0.01 M phosphate-buffered saline (PBS), pH 7.4, containing the following secondary antibodies: goat anti-mouse IgG labeled with Alex Fluor 488 or goat anti-mouse IgG labeled with Alexa Fluor 594 . Finally, the nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). To provide negative controls, we performed staining in the absence of the primary antibodies by adding only PBS to the sections. Sections were documented with a fluorescence microscope .Sections were blocked in 10 % goat serum for 30 min at 37 °C. For double labeling, two compatible primary antibodies were simultaneously applied overnight at 4 °C. The following primary antibodies were applied: mouse monoclonal anti-BrdU antibody  and rabbit anti-After the rats were sacrificed , the skin from the dorsum of each rat was taken. After that, the skin tissue was successively rinsed, immersed, digested, and incubated. After peeling off the epidermis and cutting into the microskin, the skin sample was digested with 0.25 % trypsin  to prepare a single cell suspension, which was stopped with the addition of an equal volume of high-glucose Dulbecco’s modified Eagle’s medium (DMEM). After filtering and removal of the supernatant of the cells, the cells were resuspended in high-glucose DMEM. After 15 min at 37 °C, the above ones were detected under an inverted phase-contrast microscope. The suspended cells were collected besides the adherent cells. After 24 h, the culture medium of the remaining cells was changed to keratinocyte serum-free medium (K-SFM) . When the culture reached 70 % to 80 % confluence, the cells were digested in 0.25 % trypsin at 37 °C with 5 to 10 min of oscillation and passaged at a ratio of 1:2.2 in K-SFM. The medium of the cells was supplemented with either Jagged1/FC  or DAPT for interfering with the action of the Wnt/β-catenin and Notch signaling pathway. Cells in the medium with PBS were used as the control group.ESCs were plated in six-well plates at a density of 10,000 cells/cmc-Myc antibody (Bioss), rabbit anti-Jagged1 antibody (Bioss), rabbit anti-Notch1 antibody , and rabbit anti-Hes1 antibody (Bioss). Immunoreactive bands were visualized by peroxidase-conjugated secondary antibodies. The signal from immunoblotting bands was captured  and quantified by using GIS1000 software. Quantitative Western blot measurements of target protein were normalized by corresponding measures of GAPDH derived from the same samples in each blot.Proteins of cell lysates or the skin homogenates were separated by 5 % SDS-PAGE electrophoresis and transferred into nitrocellulose membranes. After blocking with TBS/5 % nonfat dry milk, the membrane was incubated with the following antigens: rabbit anti-Wnt-1 antibody , rabbit anti-β-catenin antibody , rabbit anti-t test. The differences between the groups at different time points were compared by one-way analysis of variance followed by the Bonferroni test. All statistical analyses were performed by using SPSS 20.0 software , and a P value of less than 0.05 was considered statistically significant.Data are expressed as the mean ± standard error of the mean. Comparisons of changes in levels of signaling component expression between control and experimental groups at the same time point were conducted by using Student’s c-Myc, Notch1, Jagged1, and Hes1, as Wnt/β-catenin and Notch signaling components, were examined by Western blot to evaluate the action of two signaling pathways during the wound repair process. As shown in Fig. P < 0.05, Fig. To achieve the gain- and loss-of-function studies of Wnt/β-catenin and Notch signalings during wound healing, LiCl, DKK1, rhNF-κB, and DAPT were respectively applied to the wounded skin. The disturbing reagents above were topically and respectively applied to the wound under the transparent semi-occlusive dressing (3M) once daily until wound closure in experimental groups, allowing the solution to bathe the wound. In the control group, there was no treatment. Wnt1, β-catenin, P <0.05, Fig. Similarly, when comparing relative protein values of Notch signaling components between the rhNF-κB/DAPT group and the control group, the values in the rhNF-κB group were much higher than that in the control group, and that in the DAPT group was significantly lower at the above time points .The roles of Wnt/β-catenin and Notch signalings in wound healing were first investigated by comparing the rate of dermal wound repair in all groups. Full-thickness skin wounds were made on each side of the dorsum of the rats and were randomly divided into the experimental groups  and the control group. Wounds were observed until closure or animals were killed and were digitally photographed at the time of generation (day 0) and again on days 7, 14, 21, and 30 or until wound closure. The residual wound area rate was calculated as [(day n area) / (day 0 area)] × 100 %  by targeting Hes1 [As we all know, ESCs have an important role in wound repair , 24 and feration . The putll cycle  and shore [c-Myc . It is we [c-Myc . A possiing Hes1 .c-Myc overexpression could promote oriented differentiation of ESCs [c-Myc knock-out mice display hindered re-epithelialization, because of the inability of ESCs to produce daughter keratinocytes [c-Myc, promoting ESC differentiation into keratinocytes [Hes1 can promote senescence and differentiation of NHOKs  [To assess the ability of Wnt/β-catenin and Notch signaling in the regulation of keratinocytes, we carried out H&E staining of the lesions at several specific times after wounding. Histological analysis of the lesions reveals Fig.  that aug of ESCs . Severalinocytes . Hence, inocytes . Howeverinocytes , 33. Furinocytes . Accordinocytes) .c-Myc significantly restrains the development of anagen hair follicles, whereas c-Myc activation promotes hair growth [c-Myc expression was also observed in neoformative hair follicle cell nucleus of wounded skin (Fig. c-Myc. Additionally, Notch1 is involved in regulating invagination of hair follicles into the dermis and maintaining postnatal hair homeostasis, which indicate that Notch signaling in the wound-healing process is required for hair follicle regeneration [Hes1 in the resting phase but express high levels in the anagen phase follicles [Hes1 expression in neoformative hair follicles and its upregulation tightly following Notch activation (Fig. Hes1.Although the loss of an adult follicle is considered permanent, full-thickness excisional wounds in rats were found to heal with de novo hair follicle regeneration and this process requires the involvement of Wnt/β-catenin and Notch signalings in our results Fig. . Indeed,r growth . In thiskin Fig. . Thus, wneration . Hair foollicles . Consideion Fig. , we hypoHes1 [Hes1.In the proliferative phase of wound healing, fibroblasts produce ECM required by closing the lesion. Among this, collagen is a key component of ECM . ECM actHes1 . Taking Hes1 (Fig. Based on the gain- and loss-of-function studies of Wnt/β-catenin and Notch in the mouse models Fig. , it is les1 Fig. . It has This report provides new evidence for the potential function of Wnt and Notch signalings in regulating wound repair and their interaction. But our work has just begun. The communication among them is complex, and the mechanism that mediates Wnt and Notch pathways remains unclear. Further research is needed to elucidate the mechanism of Notch/Wnt interaction in cutaneous repair, which allows the development of new therapeutic strategies for delayed healing and pathological scarring.


Micro-RNAs are a novel class of biomarkers that have the potential to represent biomarkers of disease progression. In this study, the onset and progression of diabetes was followed in ZDF rats from six weeks until 17 weeks of age. After an initial phase of hyperinsulinemia, the animals developed T2D and lost the capacity to produce sufficient insulin. Circulating miRNAs were measured from plasma samples at four time points: pre-diabetes (six weeks of age), hyperinsulinemia (eight weeks), β cell failure (11 weeks) and late-stage diabetes (17 weeks) using TaqMan miRNA arrays. Bioinformatic analysis revealed distinct changes of circulating miRNAs over time. Several miRNAs were found to be increased over the course of the disease progression, such as miR-122, miR-133, miR-210 and miR-375. The most significantly decreased miRNAs were miR-140, miR-151-3p, miR-185, miR-203, miR-434-3p and miR-450a. Some of the miRNAs have also been identified in type 2 diabetic patients recently and, therefore, may have the potential to be useful biomarkers for the disease progression of T2D and/or the treatment response for anti-diabetic medications.The aim of the present pilot study was the identification of micro-RNA changes over time during the development and progression of type 2 diabetes (T2D) in Zucker diabetic fatty rats (ZDF rats). T2D is a complex metabolic disorder that is characterized,  Type 2 diabetes (T2D) affects 285 million people worldwide 2010), and the numbers are projected to increase substantially in the years to come up to 366 million in 2030 0, and th. In ordeob/ob mice [et al. [Micro-RNAs (miRNAs) are small, 18–22 nucleotides in length, non-coding RNA molecules that modulate the differentiation, growth, apoptosis and proliferation of cells by interfering with protein synthesis by either inducing mRNA degradation or repressing translation ,6. Circu/ob mice . There a/ob mice . Defecti [et al. .ob/ob mice [db/db mice [Alterations of miRNAs within the islets have also been reported in many animal models, such as obese models for T2D using /ob mice , db/db m/db mice  and high/db mice  or the n/db mice  and the /db mice . Mostly,/db mice , and miR/db mice .Despite the fact that type 2 diabetes is a complex disease, which is strongly influenced by the lifestyle of Western societies with high caloric diets and reduced physical activity, as well as underlying genetic factors, it seems that certain miRNAs are clearly associated with type 2 diabetes, and further research is required to understand the causes and consequences.The Zucker diabetic fatty (ZDF) rat model has become a gold standard to monitor disease progression of T2D, as many characteristics of human conditions are reflected . The ZDFIn the present study, we aimed to identify changes in the plasma miRNA profile of ZDF rats during the natural progression of T2D. Most of the altered circulating miRNAs reflect changes in diabetes-related tissues, such as pancreas, liver, heart, skeletal muscle and adipose tissues. These miRNAs could potentially serve as prognostic markers of disease progression and potentially as treatment effect markers in T2D.n = 10). The body weight of ZDF rats increased from 6–12 weeks of age and reached a plateau around 12 weeks  revealed that the overall expression profiles differed among the four different time points of disease progression . In contp < 0.05)  . At init < 0.05) . When β  < 0.05) . miRNAs, < 0.05) . At latees stage . Two miRes stage .F-statistic p-value < 0.05)  . Overall < 0.05) . Overall < 0.05) . The blo < 0.05) . The pro < 0.05) . The dro < 0.05) , and theThe present study characterizes the changes in the free circulating level of micro-RNAs in blood during the natural progression of T2D in chow-fed, aging ZDF rats from pre-diabetes over initial hyperinsulinemia to β cell failure and late-stage diabetes. The rats show initial hyperinsulinemia at eight weeks of age to compensate for the insulin resistance. Then, the insulin secretion declined until 11–12 weeks of age, when the β cells finally fail to secrete sufficient amounts of insulin and the animals become fully hyperglycemic and diabetic, also represented by HOMA-β. During the progression of T2D, the numbers of significantly altered miRNA species are constantly increased from two miRNAs at HI (week 8 of age) to five miRNAs at BCF (week 11 of age) to 12 miRNAs at LSD (week 17 of age), indicating physiological responses to diabetic complications.The circulating level of 10 miRNAs displayed significant alterations during the progression of the disease . In geneMost of our identified plasma miRNAs are known to be modified in tissues known to play a role in diabetes, such as pancreas, liver, skeletal muscle and adipose tissues or in the cardiovascular system.Based on previous work, the expression of the two increased miRNA species miR-375 and miR-210 and the three decreased miRNAs, miR-203, miR-185 and miR-450a, is involved in the regulation of insulin secretion and pancreatic β cell function ,26,27,28db/db mice and in mice fed with a high fed diet [db/db mice and high fat diet-fed mice (HFD mice) compared to our study. The study of Nesca et al., 2013, observed a decrease in miR-210 in pancreatic islets [miR-203 levels were strongly decreased at all time points during the progression of the disease. Therefore, miR-203 might be considered as an early indicator of type 2 diabetes onset in ZDF rats. In a recent study, decreased miR-203 and miR-210 expressions were detected in pancreatic islets of young pre-diabetic and diabetic fed diet  and werefed diet . Therefoc islets . Our stuc islets .Furthermore, our study showed significant alteration in the circulating level of miR-185 and miR-450a during the progression of the disease. While the role of miR-450a on the regulation of endocrine function or in diabetes is not clear to date, miR-185 plays an important role in the regulation of insulin secretion and β cell growth via targeting suppressor of cytokine signaling 3 (SOCS3) . A signiThe miR-133a displayed the strongest elevation during disease progression in our study. miR-133a is abundantly expressed in skeletal muscle and myocardial cells ,35. OverThe miR-151-3p level was decreased at late-stage diabetes in ZDF rats. In a recent study, downregulation of miR-151-3p was demonstrated during later stages of transverse aortic constriction-induced cardiac hypertrophy in mice, reflecting its participation in heart dysfunction . In our At β cell failure, circulating miR-434-3p levels were strongly decreased. Due to high variability at HI, no conclusion can be drawn about the early time point. In aged mouse skeletal muscle, miR-434-3p levels were found to be decreased  and in AOur miRNA profiling study revealed that miR-122 expression is increased in plasma of ZDF rats during disease progression of T2D. miR-122 is considered to be a highly sensitive and specific biomarker in blood, reflecting hepatocyte injury, as increased levels of miR-122 were described in various liver diseases, such as non-alcoholic steatohepatitis (NASH) . In NASHThe strongest diminished miRNA in blood was miR-140, reaching an approximately 12-fold reduction at late-stage diabetes. Decreased miR-140 levels are associated with impaired regulation of osteogenesis and adipogenesis . AdipocyIn recent years, many different profiling studies in humans and animal models have used various profiling platforms to detect miRNAs as potential biomarkers for the disease progression of type 2 diabetes. There are large numbers of altered miRNAs obtained from cultured cells, blood or tissues, which described potential early candidate markers of T2D ,49,50. TThe assessment of circulating miRNAs has several analytical and technical challenges. Plasma samples can be contaminated with cell remnants form erythrocytes, leukocytes and platelets. Erythrocytes have high levels of miR-16 and miR-451, which correlate to the degree of hemolysis in plasma samples . In partOne limitation of the current study is that it does not include a ZDF lean control group to control for potential age-related miRNA changes in non-diabetic rats. We acknowledge the possibility that age-related changes could impact miRNA levels in plasma. However, it has to be taken into account that the life span of a normal rat is 1.5–3 years (78–156 weeks). In ZDF rats, the onset of diabetes occurs at a very young age, around 8–12 weeks. In our study, the rats were used until an age of 17 weeks, which corresponds to a young age in normal rats.In principle, several reports have implicated miRNAs in cellular senescence and aging. Mostly, these changes were observed on tissue level . The folNone of the above mentioned miRNAs were found to be significantly changed in the plasma of diabetic ZDF rats in the age range of 8–17 weeks in the current study. Taken together, it is not expected that the observed changes in plasma miRNAs in diabetic ZDF rats can be explained by senescence and aging.The treatment effects of anti-diabetic drugs on miRNA expression are well described. Metformin leads to decreased circulating miR-140-5p (miR-140) and miR-122 levels in T2D patients . In endoThis pilot study provides a first insight into the altered circulating miRNA profile during the disease progression of ZDF rats, and the changes in the miRNA profile likely reflect pathophysiological complications in several tissues, such as pancreas, liver, skeletal muscle, cardiac muscle and adipose tissue. Further studies are needed to underline their potential as early treatment effect biomarkers, as previously described for miRNA-29s. These novel, non-invasive markers show promise as tools for the mechanistic investigation of early disease progression of T2D and potentially to monitor treatment effects. As miRNAs regulate a number of downstream targets, the identified changes may be able to additionally uncover disease mechanisms in the ZDF rat. Future studies should elucidate the functional implications of the observed plasma miRNA changes for each target tissue.fa/Crl  were housed in groups of 2 under pathogen-free conditions, controlled temperature on a regular 12-h light-dark cycle and received standard chow (Harland 2018) and water ad libitum. Animal experiments were conducted at the Centre Hospitalier de l´Universitć de Montréal and in accordance with the Policies and Guidelines of the Canadian Council for the Protection of Animals. The study code was BTX-BO002. All animals had an acclimation period of 5 days prior to the study start. Animals in the study were from 6 weeks of age until 17 weeks of age (n = 10). Body weight, 2-h-fasted glycemia, insulinemia and HbA1c were measured at baseline and then weekly from plasma or blood samples. For this, blood was taken from the tail vein of non-anaesthetized rats. All blood draws were carried out at the same time of the day following a 2-h food deprivation. Fasting glucose and fasting insulin were used to calculate the homeostasis model assessment of β cell function (HOMA-β). HOMA-β was calculated as (20 × fasting glucose (μU/mL)/fasting glucose (mmol/L) − 3.5).The longitudinal T2D progression study was performed at Bêtagenex in Laval, Québec, Canada. Male ZDF Leprn = 3 pooled samples per group) was performed using the miRNeasy Serum/Plasma Kit .For the micro-RNA discovery analysis, pooled plasma from the ZDF rat samples was investigated. In detail, three plasma samples from the pre-diabetes group (PD) (6-weeks-old ZDF rats) and from animals aged 8, 11 and 17 weeks were used. Pooling was done according to insulin levels at baseline in a manner that reflects the overall mean in each pool. miRNA extraction of 150 μL pooled plasma  according to the manufacturer’s protocol. With content matched to Megaplex stem-looped Rodent RT primers, Megaplex PreAmp Rodent Pool A Primers are used for unbiased preamplification, as recommended by the vendor.miRNA samples were normalized to 3.5 ng/μL and reverse-transcribed using the Megaplex Primer Rodent Pool A and the Taqman® Fast Advanced MasterMix (Applied Biosystems) and the rodent Taqman® miRNA Array, Card A V.2.0 (Applied Biosystems). The gene expression analysis was run on a SDS7900HT real-time PCR system (Applied Biosystems by Thermo Fisher Scientific); raw Ct (cycle threshold) values were calculated using the SDS software v2.4 (Applied Biosystems by Thermo Fisher Scientific) with automatic baseline and threshold settings. Data were normalized to global mean Ct. miRNAs with Ct values >32/undetermined were excluded. At least two of three replicates had to fulfill this criterion to be considered in the relative expression computation  [The expression of the miRNAs was screened using Taqman method) .Ct < 35 for global mean calculation. For further analysis, only those miRNAs with raw Ct < 32 in at least two samples of at least one group were considered. For differential expression analysis, limma v3.18.13 [versus baseline (week 6), week 11 versus baseline, as well as week 17 versus baseline are reported. p-values are the false discovery rate (FDR) adjusted as proposed by Benjamini and Hochberg [F-statistic p-value ≤0.05 are considered significantly altered in the ANOVA . The Bioconductor package HTqSeq v1.16.0  was usedv3.18.13  was applHochberg . miRNAs he ANOVA . For groue ≤0.05 .


Background: To report the clinical outcome after a single implant, high dose rate (HDR) brachytherapy in early prostate cancer.Materials and Methods: All clinically localized prostate cancer patients who underwent high-dose rate (HDR) brachytherapy as monotherapy  from February 2006 to September 2011 were analyzed prospectively. Acute and chronic toxicity were assessed as per Common Terminology Criteria for Adverse Events (CTCAE), Version 4.03. Biochemical recurrence was analyzed using the Kaplan Meir method. A log-rank analysis was done to compare the factors affecting the outcome.Results: Forty-four patients with organ-confined prostate cancer opted for HDR brachytherapy between February 2006 to September 2011 with a median follow-up of 68 months  The five-year biochemical recurrence-free survival (bRFS) rate was 91%. Late Grade 2 genitourinary (GU) toxicity was observed in 9% of patients. The predictors of late Grade 2 GU toxicity were urethra V125 ≥ 0.2 cc  and PTV 150 ≥ 35% ( planning target volume receiving ≥ 150% of the prescribed dose) with p-value = 0.001 and 0.002, respectively. Erectile function was preserved in 72% of the patients who had Grade 0-1 erectile dysfunction before brachytherapy.Conclusion: HDR brachytherapy in early prostate cancer results in high local control rates with minimal side-effects. The treatment options for organ-confined prostate cancer range from radical prostatectomy to image-guided radiotherapy, brachytherapy, protons, stereotactic body radiotherapy (SBRT), and high-frequency ultrasound (HIFU). Local expertise, equipment available, and physician bias often dictates which modality is advised. In spite of brachytherapy being recognized as one of the most conformal forms of radiotherapy, the gaining popularity of intensity modulated radiotherapy, protons, and SBRT has led to a gradual decline in the practice of brachytherapy in the developing world. High-dose rate (HDR) brachytherapy is a minimally invasive procedure to deliver highly conformal radiotherapy in a very short time with minimal side-effects. A few groups have used it in a monotherapy setting with excellent results -9. The aThe Apollo Hospital Educational and Research Foundation Ethics Committee issued approval of this prospective study and waived informed patient consent.www.nccn.org) which defines low-risk as PSA ≤ 10 ng/ml, T1c-T2 and a Gleason score (GS) ≤ 6; intermediate risk as PSA 10-20 ng/ml, or GS 7; and high risk as a PSA > 20 ng/ml, T3, or GS 8-10. The inclusion criteria were an intact capsule with any Gleason score or PSA. Exclusion criteria were radiologically positive lymphadenopathy, distant metastasis, and prior pelvic radiotherapy. Intermediate and high-risk patients were offered hormonal treatment. Ten out of the fourteen high-risk patients opted for concurrent hormonal therapy. None of the low and intermediate risk category patients received hormonal therapy.Forty-four patients with organ-confined, histologically proven adenocarcinoma of the prostate gland underwent HDR monotherapy during February 2006 to October 2011 after informed patient consent at the time of their treatment. Digital rectal examination, prostate-specific antigen (PSA), computerized tomography (CT), magnetic resonance imaging (MRI) of the pelvis, and a Technetium-99 bone scan (in high-risk patients) was mandatory. Risk stratification was done as per the National Comprehensive Cancer Network (NCCN)  is mounted. The template is jammed against the perineal skin. There is a grid faceplate fixed onto the template, which corresponds with the grid of the TRUS for accurate placement of the ProGuide needles. Under TRUS guidance, metallic trocars are inserted transperineally through the holes in the template to ascertain the position in the prostate. Then the trocars are removed and replaced by the 6F ProGuide plastic needles in the same position. We always start with the peripheral and anterior needles first and then move towards the center. As far as possible, the needles are placed at 1 cm intervals. No needles are placed within 7 mm of the urethra to have a control on the urethral dose. The needles are pushed beyond prostate base into the bladder mucosa. The posterior needles are placed 2-3 mm anterior to the anterior wall of the rectum to avoid overdosing rectum. The template has a seamless single action locking system for preventing any inadvertent outward movement of the needles. It is immobilized by stitching the four corners of the template onto the skin. Once the needles are in position, a planning spiral CT scan in the supine position is done with 1 mm slice thickness and images are transferred to the Nucletron brachy planning system.The planning target volume (PTV) is contoured by the radiation oncologist on each CT slice and includes the prostate with a 3 mm margin all around except posteriorly where no margin is given to avoid overdosing the anterior rectal wall. Superiorly, a margin of 5-7 mm is given to compensate for any post-implant edema and inadvertent caudal movement of the catheters in between the fractions. The PTV constraints were D90 (dose delivered to 90% of PTV) ≥ 97%, V95 ≥ 100%, and V150 ≤ 35% , Version 4.03. Erectile dysfunction (ED) was scored as follows: Grade 0 = no symptoms of ED, Grade 1= decreased erectile function but able to perform intercourse, Grade 2 = decreased erectile function not able to perform intercourse, and Grade 3 = no erection.We also recorded the pre- and post-treatment IPSS  to evaluate the impact of treatment on IPSS scores. The IPSS is based on answers to seven questions about urinary symptoms  and one question concerning the quality of life. The patient assigns a score of 0 - 5 for the urinary symptoms and 0 - 6 for assessing the quality of life due to the severity of urinary symptoms. The total score can range from 0 to 35 (asymptomatic to very symptomatic). A 1-7 score is graded as mild, 8-19 moderate, and 20-35 as severe.Phoenix criteria were used for assessing biochemical recurrence-free survival (bRFS) that defined biochemical recurrence as a PSA rise of 2 ng/ml above the nadir PSA.Continuous variables were assessed for normality using Shapiro Wilk’s test. The variables, which followed the normal distribution, were expressed either as mean + SD or median (interquartile range). All other categorical variables were expressed either as a percentage or proportion. Comparisons of categorical variables were done by either Chi-square test or Fisher’s exact test based on the number of observations. Pre-brachytherapy and post-brachytherapy comparison of normally distributed continuous variables like PSA and IPSS were done by Paired ‘t’ test.  Biochemical recurrence-free survival rates were calculated using the Kaplan-Meier method. The duration of follow-up for bRFS was calculated from treatment to PSA relapse as defined by the Phoenix criteria, the development of distant metastasis, or to the last follow-up. Cancer-specific survival was not analyzed, as there was only one cancer-related death. The log-rank test was done to compare the effect of age and risk stratification on the bRFS outcome. Data entry was done in an MS EXCEL spreadsheet. Data validation and analysis was performed by SPSS V16.0. Statistical significance was considered on a two-sided significance level (α) of 0.05.Patient and dosimetric characteristics are given in Tables The mean IPSS was 14 before brachytherapy and reached a peak mean of 18 twelve weeks post-brachytherapy Figure . IPSS reThe most common acute symptoms post-brachytherapy were frequency and urgency. Thirty-six percent and 28% of the patients had Grade 1 and 2 frequency, respectively. Grade 1 urgency was seen in 18.6% and Grade 2 in 11.6% patients. Sixteen percent of the patients had hematuria after removing the needles. Seven percent of the patients went into retention that was related to blood clots causing obstruction, necessitating re-catheterisation for a couple of days. No patient experienced Grade 3 or 4 acute GU toxicity. Fifteen percent of the patients had Grade 1 gastrointestinal (GI) symptoms of tenesmus and pain while defecating. No patient had Grade 2, 3, or 4 GI symptoms  and PTV 150 ≥ 35% (planning target volume receiving ≥ 150% of the prescribed dose) with the p-value = 0.001 and 0.002, respectively (Table Five patients had Grade 2-3 erectile dysfunction (ED) before brachytherapy. Of the remaining thirty-nine patients having useful sexual intercourse, eleven patients progressed to Grade 2-3 ED after brachytherapy. Twenty-eight of 39 (72%) of patients were able to perform sexual intercourse.In the thirty-two patients who did not receive hormonal therapy, the median PSA nadir was 0.75 ng/ml. PSA bounce was defined as a rise in PSA of greater than 0.2 ng/ml or higher, which came down to at least the same level or a lower level. The median duration to achieve the nadir was 36 months. Eleven patients (34%) had a PSA bounce. The median amplitude of the bounce was 0.5 ng/ml. This happened at a median of 18 months after brachytherapy and lasted for a median of six months before the PSA started falling again  after univariate analysis. There are at least 13 groups who have published their HDR monotherapy data with a median follow-up ranging from 1.5 years to 5.4 years. Though comparison with these groups is difficult because of the considerable variation in the dose and fractionation schedules, the five-year biochemical control rates reported vary from 85-97% for low-risk, 93-97% for intermediate risk, and 79-88% for high-risk disease in organ-confined prostate cancer [Most of our patients were treated with a single implant with 3 fractions of 10.5 Gy each. Hoskin, et al. [3 of the rectum). They did not find any dosimetric parameter predicting urethral toxicity.Yoshioka  reportedMark, et al.  and RogeIn our group, the multivariate analysis using Kaplan-Meir actuarial estimates of bRFS did not show any statistically significant association with age, Gleason score, initial PSA, or clinical stage. Rogers, et al. and Hoskin, et al. also failed to find any significant association of biochemical relapse with clinical stage, Gleason score, or pre-treatment PSA. In the Yoshiaka, et al. series, the significant prognostic factors for PSA failure were the initial PSA level (p = .029) and younger age (p = .019).All the groups have consistently reported excellent long-term control rates with minimal GI/GU morbidity across various fractionation schedules doses and risk groups.The main limitation of this study is the small number of patients. Another limitation was although the data was collected prospectively, the analyses were done retrospectively. Our study adds to the already existing evidence for the effectiveness of HDR monotherapy in clinically localized prostate cancer.HDR brachytherapy as monotherapy for organ-confined prostate cancer results in high biochemical control rates with minimal side-effects.